Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shear-based procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by mass spectrometric (LC/MS/MS) analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G Myo1G). Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament- and microtubule-related proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells. We have chosen for intensive investigation two of more abundant molecules identified as strongly enriched in the membrane/microvillus fraction in the foregoing studies: Myo1G and NHERF1. Myo1G was chosen because class I unconventional myosins in other cells have been shown to play important roles in regulation of microvilli formation, endocytosis, vesicular traffic and other key cellular events but their role in hematopoietic cell function is largely unstudied. Myo1G in particular is completely unstudied, despite its prominent expression in T-cells. We have therefore have initiated systematic investigation of Myo1G. Key reagents have been generated including a specific polyclonal antibody, constructs to study structure-function relationship and a conditional knockout mouse These tools are being used to investigate Myo1G expression, localization, and function in cells and in the mouse. Similarly, NHERF1 is a PDZ-containing adaptor molecule important in regulating molecular localization and function in multiple cells types, but whose function is unstudied in the immune system. We have initiated a collaboration to study knockout mice, and are back-crossing the mouse strain to Bl6 to improve their usefulness for immunologic analysis. Initial results indicate that knockout lymphocytes have alterations in cell spreading and migration. Systematic analysis is underway.